This document discusses various diagnostic techniques for viral animal diseases. It describes direct detection methods like electron microscopy, histopathology, and fluorescent antibody techniques. It also covers indirect detection methods like ELISA, immunochromatography, latex agglutination, and viral antibody detection techniques like complement fixation, haemagglutination inhibition, and virus neutralization tests. Emerging techniques discussed include PCR, microarrays, and nanobiosensor-based diagnostics.

1. Viral diagnostics for animal
diseases
I.Sophia
P-1699
VBM

2.  substances or devices which aid in the diagnosis of viral infections
 Classification of viral diagnostic tests
 (i) direct virus detection
 (ii) viral antigen detection
 (iii) virus isolation
 (iv) viral antibody detection.
Viral diagnostics

3.  Electron microscopy, immunoelectron microscopy
 Histopathology
 Fluorescent antibody technique – direct, indirect
 Immunohistochemistry
 ELISA
 Immunochromatography
 Latex agglutination test
Direct identification of viruses

4.  ELECTRON MICROSCOPY
 Rapid diagnosis
 No need of additional probes
 Disadvantage
 Procurement and maintenance of the instrument
– expensive
 Require skilled & well trained personnel for
operation.
 Sensitivity is comparatively low – require at least
10 6 viral particles for + ve diagnosis.
 Single specimen examined at a time.
 Applications:
 Rotavirus from faecal samples, rabies virus from
brain, norwalk virus from faecal samples

5.  Employs viral antibody
 Increases the sensitivity & specificity of EM
 Two methods (i) classical IEM (ii) solidphase IEM
 classical IEM: Requires prior incubation of the clinical sample with virus
specific antibody .
 Solidphase IEM: solid support (copper grid) is coated with specific antibody
which captures viruses from the clinical sample.
Immunoelectron microscopy (IEM)

6.  Helps in diagnosis of viral inclusion bodies
 Viral inclusion bodies: Aggregates of viral nucleocapsids in the cytoplasm or
nucleus.
 Can be detected by H&E staining
Applications
 Negri bodies in rabies virus
 Guarnieri bodies – vaccinia virus
 Bollinger bodies - fowlpox
Histopathology

7.  Use of two specific antibodies – nanoparticle conjugated antibody, antibody
fixed in chromatography paper.
 The sample dropped on sample pad – forms complex with the antibody. When
moves along the membrane pad- sandwiched between two antibodies-
production of colour.
 diagnosis of avian influenza, dengue etc.
IMMUNOCHROMATOGRAPHY

8.  Particulate antigen +virus specific antibody - crosslinking of polyvalent
antigens – agglutination.
 Visibility can be improved by coating the latex beads with virus specific
antibodies (sensitized latex beads).
 Sensitized latex beads + clinical sample (containing viruses) visible
aggregates.
 Applications:
 Avian influenza
 Rota virus
 FMD (Sugimura et al.,2000)
Latex agglutination test

9. Fluorescence antibody technique (FAT)
 Fluorochromes like fluorescent isothiocyanate and rhodamine isothiocyanate. Are
used.
 Viral antigens can be detected from smears from clinical samples, frozen tissue
sections and also from formalin fixed tissue samples.
Direct Fluorescence antibody technique
The virus specific antibodies are directly tagged with fluorescent dyes.
Indirect fluorescence antibody technique (IFAT)
second antibody known as anti-species immunoglobulin (Igs) are used.
Advantage:
 Available easily commercially than the virus specific antibodies
 More sensitivity than direct FAT.
 Better signal amplification

10. Immunohistochemistry
Principle
 Employs antibodies conjugated with enzymes like horseradish peroxidase or
alkaline phosphatase.
 When suitable substrates are used, colour reaction takes place in case of
positive sample which can be viewed through light microscope.
 Both direct and indirect immunoperoxidase tests are used.
 Advantages
 The procedure can be applied for formalin fixed tissues as well.
 Comparative study of site of localization of viruses and the tissue damage is
possible

11. Sandwich ELISA
The viral antigen is sandwiched between two antibodies namely capture and
detection antibodies. For the assay both the antibodies should target different
epitopes of the antigen. This is followed by addition of enzyme conjugated anti-
species immunoglobulin. On adding suitable chromogen-substrate colour
reaction develops.
Advantages
Assay is quantitative, amount of viral antigen can be detected
Assay has high sensitivity and specificity
More samples can be tested at the same time
Disadvantages
Need ELISA reader for result interpretation; not possible under field conditions
The method is time consuming and labourious.
Applications:
Used in diagnosis of PPR, Bluetongue, FMD etc

12. Immunoblotting
Applications: useful in differentiation of infected and vaccinated
animals in FMD using recombinant proteins (Fu et al., 2011).

13. Complement fixation test
Principle
 The ability of the complement system to fix the antigen- antibody complex
forms the basis of complement fixation test.
 Sheep RBCs and its corresponding antibody acts as indicator system.
 When the antigen reacts with specific antisera, antigen antibody complex will
be formed and the complement will be unavailable for the indicator system.
Then there will be no haemolysis and the test is positive.
 If the antiserum is not specific, then the complement is free to fix the
indicator system resulting in haemolysis.
 Sensitive assay like ELISA have replaced it.
 Simple; easy to perform.
Applications
 Complement fixation test is prescribed by the OIE for diagnosis of equine
diseases like African horse sickness, equine encephalomyelitis (eastern,
western, Venezuelan), vesicular stomatitis.

14. Haemagglutination inhibition tests
Principle
Certain viruses namely paramyxoviruses and orthomyxoviruse have the ability to
bind to sialic acid residues in RBCs. This property is called haemagglutination.
If specific antibody and viruses are mixed prior to the addition of RBCs
haemagglutination is inhibited.
HI test is used for serotyping and also for measuring antibody titre.
Applications
 OIE recommends this test for diagnosis of viral infections like blue tongue,
avian laryngeo tracheitis, avian influenza, marek’s disease, infectious bursal
disease, enzootic bovine leucosis, equine infectious anaemia (Coggin’s test),
myxomatosis and caprine arthritis encephalitis disease.

15. Complement fixation test
-ve
+ve

16. Virus neutralization test
Principle
 When a serum sample to be tested is specific for the virus, there will be
antigen-antibody complex formation. The virus particles are unavailable to
cause cytopathic effect in cell culture which can be visualized microscopically
or by staining with vital dyes.
Applications
 Prescribed test by OIE for diagnosis of pseudorabies, rabies, Riftvalley fever,
vesicular disease and IBRT-IPV.

17. Plaque reduction neutralization test
Principle
The ability of virus specific antibody to inhibit the plaque forming property of
viruses by 50% when plated in semisolid media is the basic principle involved.
Applications
PRNT is recommended by the OIE for diagnosis of eastern equine encephalitis
and Venezulean equine encephalitis disease.
Disadvantages:
The interpretation of results requires at least 3-5 days which is very slow when
compared to EIA and RIA.
Both the tests involve use of cell culture where false results due to
contamination of cell culture are possible.

18. Agar gel immunodiffusion test
Principle
 It is basically a precipitation test in which soluble antigen reacts with its
homologous antibody in the presence of electrolytes. At the optimum
proportion, an insoluble antigen-antibody precipitate will be formed.
Applications
 OIE recommends this test for diagnosis of viral infections like blue tongue,
avian laryngeo tracheitis, avian influenza, marek’s disease, infectious bursal
disease, enzootic bovine leucosis, equine infectious anaemia (Coggin’s test),
myxomatosis and caprine arthritis encephalitis disease.

19. Counter immunoelectrophoresis
PRINCIPLE
Counter immunoelectrophoresis is the immunodiffusion modified by
electrophoresis to drive antigen and antibody towards each other. The specimen
to be tested is placed in the cathode side, and the antiserum is placed in the
anode. At neutral or alkaline pH, antigens are negatively charged and hence
migrate towards anode in barbital or veronal buffer.
Applications:
Counter immunoelectrophoresis is used in diagnosis of blue tongue virus, orf
virus and pox viral diseases

20. Radio immunoassay
Principle
It is the competitive binding assay which binding of known amount of
radiolabeled substrate and antibody. When the test sample is added, the
radiolabeled substrate gets displaced by it resulting in free radiolabelled
substrate in the solution. Finally, the radioactivity of free substrate in the
solution is measured which is proportional to the amount of unknown antigen
bound to antibody.
The assay is more sensitive and specific compared to the above methods.
Disadvantage:
Assay needs sophisticated instruments like gamma counter.
Radioactive compounds are hazardous to human health ie. Carcinogenic.
Therefore, it is not routinely used for viral diagnosis.

21. Indirect ELISA
PRINCIPLE
 The viral antigen is immobilized in a solid support followed by addition of
serially diluted antibodies. The antibodies in turn are captured by enzyme
conjugated anti-species immunoglobulin.
 Addition of chromogen / substrate facilitates the colour reaction which can
be measured calorimetrically.
 Currently, use of recombinant viral proteins as antigens reduce the risk of
handling of dangerous organisms and made the assay sensitive and specific.
Advantages
Simplicity; easy to perform
Rapid than neutralization assays
Safer: Use of enzymes instead of radiolabels
Require ELISA reader for result interpretation

22. Nucleic acid based diagnostics
 PCR
 RFLP
 Microarray
 Hybridization techniques

23. RFLP
 The nucleotide signatures of each species are unique. This fact is explored in
the technique where endonucleases recognizing restriction sites are used to cut
the genome at various sites for each organism. The genome fragments are
resolved by running in agarose gel electrophoresis. The characteristic nucleotide
base pair length corresponds to each organism.

24. PCR

25. Real time PCR

26. MICROARRAY

27. Nanobiosensor
 There are various types of nanobiosensors based on various
principles namely, electrochemical biosensors, voltametric and
amperometric sensors, impedance sensors, optical fiber based
sensors, surface plasmon resonance based biosensors, quartz
crystal microbalance and atomic force microscopy based
nanobiosensors.
 Nanobiosensors have been designed for diagnosis of dreadful
human viral diseases like HIV, hepatitis B, Hepatitis C, Ebola virus
etc. Nanobiosensors have also been designed for diagnosis of
animal diseases like avian influenza virus, infectious bovine
rhinotracheitis, rabies, bovine leukemia virus.